The goal of this project is to determine the functional role and structural importance of cardiolipin (CL) binding within two mitochondrial electron transport complexes: cytochrome c oxidase and cytochrome bc1. The two enzymes are ideal choices for probing the specific association of CL in mitochondrial electron transport complexes since both have been shown by us, or by others, to contain tightly bound CL that is essential for functional activity. The specific goals during the project period are: 1. Map and identify the subunit(s) and amino acid sequence(s) with cytochrome c oxidase and cytochrome bc1 that participate in binding CL. 2. Determine the influence of tightly bound CL on the dimerization of each complex. 3. Investigate the functional importance of bound CL upon specific individual electron transfer steps, conformational cycling, and proton translocation activity of cytochrome c oxidase. 4. Analyze the functional competence of monomeric cytochrome bc1 in proton translocation. These goals are each a logical extension of our previous studies. Each also takes advantage of a series of new experimental approaches that we have developed during the past funding period, i.e. (a) the synthesis of CL derivatives that contain a variety of functional groups on either the polar head group or apolar tail of CL; (b) new sensitive HPLC and ESI/MS methods for the analysis of the subunit content and molecular masses of each subunit; (c) steady state kinetic methods and a working kinetic model that enables us to determine the functional role of CL in cytochrome c oxidase; and (d) new analytical ultracentrifuge facilities for accurately measuring the self-association of each complex. With our unique CL analogues, approaches and methods, we will be able to map the CL binding sites within each complex, and determine mechanisms by which tightly bound CL participates or affects specific functional steps. Results obtained with these two similar, but different complexes will allow us to determine whether CL has unique or universal roles in coupled electron transport.